NMR Studies of the Secondary Structure in Solution and the Steroid Binding Site of 5-3-Ketosteroid Isomerase in Complexes

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• 作者：Qinjian Zhao ; Chitrananda Abeygunawardana ; and Albert S. Mildvan
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：March 25, 1997
• 年：1997
• 卷：36
• 期：12
• 页码：3458 - 3472
• 全文大小：546K
• 年卷期：v.36,no.12(March 25, 1997)
• ISSN：1520-4995

文摘

Backbone and side chain resonances of steroid-bound⁵-3-ketosteroid isomerase (EC 5.3.3.1),a homodimeric enzyme with 125 residues per monomer, have been assignedby heteronuclear NMR methodswith the ¹⁵N- and ¹³C-labeled enzyme. Thesecondary structure in solution of steroid-boundisomerase,based on interproton NOE's and differences in chemical shifts ofbackbone H, C, C, and CO resonancesfrom random coil values, consists of two -helices (residues 5-21,48-60), one 3₁₀ helix (residues 23-30), seven -strands (residues 34-38, 44-47, 62-67, 71-73,78-87, 92-104, and 111-116), and fiveturns (residues 39-42, 74-77, 88-91, 105-108, and 119-122).Thus isomerase consists of 30% helix,38% -sheet, and 16% turns. The remaining 20 residues (16%)are assumed to form coils. With theexception of a parallel interaction between -strands 1 and 7, all-strand interactions are antiparallel,forming both a -hairpin (1, 2) and a four-stranded -sheetin which the first strand is interrupted(3-4, 5, 6, 7). ¹H-¹⁵NHSQC titrations of the free enzyme with the substrateanalog19-nortestosterone hemisuccinate revealed steroid-induced changes inbackbone ¹⁵N and NH chemicalshifts throughout the enzyme, with maximal effects on helix 1 (Val-15),-strand 1 of the -hairpin (Asp-38), the loop between helix 3 and -strand 3 (Leu-61), -strand 3(Ala-64), -strand 5 (Phe-82, Ser-85,Glu-87), -strand 6 (Ile-98), and -strand 7 (Ala-114, Phe-116) ofthe -sheet, thus indicating the secondarystructural components involved in steroid binding. These effectsinclude regions near the catalytic residuesTyr-14 and Asp-38 which function as the general acid and base,respectively, in the ketosteroid isomerasereaction. Intermolecular NOE's between 19-nortestosteronehemisuccinate and isomerase indicate thatthe steroid binds near -helices 1 and 3, which form one wall of theactive site, and one end of thefour-stranded -sheet which forms the other wall. Consistentwith these observations, doxyldihydrotestosterone, a steroid that is spin-labeled at its solvent-exposed end[Kuliopulos, A., Westbrook, E. M.,Talalay, P., & Mildvan, A. S. (1987) Biochemistry26, 3927-3937], induced the selective attenuationinthe ¹H-¹⁵N HSQC spectra of cross peaks ofresidues at the end of helix 3 (Ser-58, Leu-59, Lys-60,Leu-61), -strand 5 (Val-84, Ser-85), and -strand 6 (Val-95), dueto the proximity of the nitroxide radicalto the backbone ¹⁵N and NH nuclei of these residues, thusconfirming the location of the D ring of thebound steroid and defining the mouth of the active site.